POS0013 25-HYDROXYCHOLESTEROL IN THE PATHOGENESIS OF RHEUMATOID ARTHRITIS
Authors: H. Huang, Y. Geng, Y. Fan, Z. Zhang
Journal: Annals of the Rheumatic Diseases (2023)

<h3>Background</h3> Rheumatoid arthritis (RA) is an autoimmune disease characterized by synovial inflammation, in which the imbalance of CD4<sup>+</sup> T cells plays an important role. 25-hydroxycholesterol (25-HC) is an important regulator of the mevalonate pathway in cholesterol metabolism. Previous studies have suggested that 25-HC may inhibit the transformation of CD4<sup>+</sup> T cells from interferon-γ (IFN-γ) <sup>+</sup>CD4<sup>+</sup> T cells to IL-10<sup>+</sup>CD4<sup>+</sup> T cells through the interaction with liver X receptor (LXR), therefore decreasing the expression of IL-10. <h3>Objectives</h3> This study aims to explore the mechanism by which 25-HC promotes the inflammatory process of RA by regulating the phenotypic transformation of CD4<sup>+</sup> T cells through histological and cellular and molecular biology techniques, and propose possible molecular regulatory pathways. <h3>Methods</h3> Patients with RA from December 2019 to January 2022, and gender and age-matched osteoarthritis (OA) and healthy controls (HC) were enrolled. Peripheral blood mononuclear cells (PBMC) and synovial fluid mononuclear cells (SFMC) were collected and CD4<sup>+</sup> T cells were sorted by magnetic beads. The expression levels of different phenotypes of CD4<sup>+</sup> T cells and important cytokines were analyzed by flow cytometry; the secretion levels of IL-10 and IFN-γ were detected by ELISA technology. In the intervention experiment, the CD4<sup>+</sup> T cells were treated with different concentrations of atorvastatin to inhibit the cholesterol metabolism mevalonate pathway and supplement mevalonate acid, and then CD4<sup>+</sup> T cell phenotype and cytokine expression were detected. The synovial tissues were obtained from RA and OA patients receiving joint replacement surgery, and the expressions of 25-hydroxycholesterol hydroxylase (CH25H) and LXR were detected by immunohistochemistry and multiplex immunofluorescence. Real-time PCR and Western Blot were used to detect the expression of inflammasome signaling pathway of CD4<sup>+</sup> T cells. CH25H was knocked down suing small interfering RNA (siRNA), and the effects of knockdown of CH25H on inflammasome and the effect on the transformation of CD4<sup>+</sup> T cells were detected by the above methods. <h3>Results</h3> (1) IL-10<sup>+</sup>CD4<sup>+</sup> T cells in PBMC/SFMC of RA patients were significantly lower than HC/OA, as well as the expression of IL-10. After interfering the mevalonate pathway by atorvastatin, the ratio of IL-10<sup>+</sup>IFN-γ<sup>+</sup>CD4<sup>+</sup> T cells and IL-10<sup>+</sup>IFN-γ<sup>-</sup>CD4<sup>+</sup> T cells as well as the expression of IL-10 decreased in a concentration-dependent manner. The down-regulation effect induced by atorvastatin was compensated after the supplementation of mevalonate acid. (2) The expressions of CH25H and LXR in CD4<sup>+</sup> T cells of RA synovial tissue increased detected by immunohistochemistry and multiplex immunofluorescence, and the expressions of CH25H, LXR and caspase-1 in CD4<sup>+</sup> T cells in synovial fluid were found increased by Western Blot, as compared with that of OA. (3) After the successful knockdown of CH25H confirmed by Real-time PCR and Western Blot, a significant decrease in the proportion of IL-10<sup>-</sup>IFN-γ<sup>+</sup>CD4<sup>+</sup> T cells, and increase in proportion of IL-10<sup>+</sup>IFN-γ<sup>-</sup>CD4<sup>+</sup> T cells were found in RA patients, accompanied with increase in IL-10<sup>+</sup>CD4<sup>+</sup> T cells, while the proportion of IFN-γ<sup>+</sup>CD4<sup>+</sup> T cells and expression of IL-10 decreased significantly. After supplementation with 25-HC, the siRNA-CH25H-mediated decrease in IL-10<sup>+</sup>CD4<sup>+</sup> T cells was reversed and IFN-γ<sup>+</sup>CD4<sup>+</sup> T cell formation was reduced. Meanwhile, the expression of NLRP3 and activated caspase-1 (capsase-1 p20) in peripheral blood CD4<sup>+</sup> T cells was reduced, and could eliminate after supplementation with 25-HC. <h3>Conclusion</h3> In peripheral CD4<sup>+</sup> T cells in RA patients, 25-HC may activate the NLRP3 inflammasome through CH25H-LXR pathway, thereby inhibiting the phenotypic transformation of IFN-γ<sup>+</sup>CD4<sup>+</sup> T cells to IL-10<sup>+</sup>CD4<sup>+</sup> T cells, and eventually promoting the inflammatory process in RA. These findings provide new clue for the mechanism of CD4<sup>+</sup> T cells in the pathogenesis of RA and suggest that the cholesterol metabolism pathway may become a new target of RA treatment. <h3>REFERENCES:</h3> NIL. <h3>Acknowledgements:</h3> NIL. <h3>Disclosure of Interests</h3> None Declared.